Amino acid derivatives of 6-aminopenicillanic acid

ABSTRACT

Novel penicillins having the formula   ARE DISCLOSED ALONG WITH METHODS FOR THE PREPARATION OF THESE COMPOUNDS.

- United States Patent Dolfini et al.

[451 Oct. 1, 1974 AMINO ACID DERIVATIVES OF 6-AMINOIENICILLANIC ACIDInventors: Joseph Edward Dolfini, Princeton;

Raymond Curry Erickson, Metuchen, both of NJ.

E. R. Squibb & Sons, Inc., Princeton, NJ.

Filed: Dec. 11, 1972 Appl. No.: 313,707

Assignee:

US. Cl. 260/239.1, 424/271 Int. Cl C07d 99/16 Field of Search 260/239.l

References Cited UNITED STATES PATENTS 11/1967 Doyle et al. 260/239.l

[57] ABSTRACT Novel penicillins having the formula are disclosed alongwith methods for the preparation of these compounds.

7 Claims, No Drawings AMINO ACID DERIVATIVES OF The novel penicillins ofthis invention are represented by the formula:

( Emma)...

wherein R ma be hydrogen, lower ikylf acylb Examples of salt formingions are the alkali metals (e.g., sodium or potassium), the alkalineearth metals (e.g., calcium or magnesium) and radicals of organic bases(e.g., dibenzylamine, N, N,- dibenzylethylenediamine).

The acyloxy group is represented by the formula Ii -(Lowherein R may belower alkyl (as defined above) or monocyclic carbocyclic aryl. Acetoxy,propionyloxy, isopropionyloxy, butyryloxy, pivaloyloxy, and benzoyloxyare specific acyloxy groups of the type contemplated by applicants.

The acyloxymethyl group may be represented by the formula R is definedin the above paragraph. Acyloxy-methyl esters of the type contemplatedfor use in this invention may be exemplified by acetoxymethyl,propionyloxymethyl, isopropionyloxymethyl, butyryloxymethyl,pivaloyloxymethyl, and benzoyloxymethyl esters. Production ofthe'acyloxymethyl esters may be achievedby a procedure adapted from themethod of Daehne et al., Jour. Med. Chem. 13, 607 (1970). A halomethylester having the formula XCH OCOR wherein R is as previously defined andX is a halogen (preferably chlorine or bromine), is reacted with either6- aminopenicillanic acid (one of the starting materials of thisinvention) or with the acidform of the penicillin of formula I.

The novel compounds of this invention (formula I). may be produced fromo-aminopenicillanic acid and the amino acids of formula II:

H 1 1 (0 2)n CHCOOH (emu-(0H2)...

wherein R, n, m, and p are as defined above. The amino acids of formulaII may be readily synthesized from known materials; alternatively,naturally occurring amino acids may be used. Aziridine-Z-carboxylicacid, azetidine-Z-carboxylic acid, proline, hydroxyproline,4-hydroxypipecolic acid, and 5-hydroxy-pipecolic acid are examples ofamino acids of the type that are useful as starting materials for thisinvention. Proline and hydroxyproline are preferred amino acids.

Reaction of an amino acid with an alkali metal cyanate or an alkalineearth metal cyanate yields an N- carbamyl amino acid (formula III):

In forming the N-carbamyl amino acid, an aqueous suspension. of theamino acid is reacted with the, cyanate. Heating the suspension for ashort period. of time (e.g., about 5 minutes) yields a solution. Thereaction proceeds at room temperature and may take anywhere from30minutes to several days, usually around 2 days. Periodically, the pHof the solution is adjusted to make it slightly acidic (i.e. a pH ofbetween about 5.0 and 6.5); the pH adjustment may be accomplished by useof a mineral acid (e.g., hydrochloric acid). The N- carbamyl amino acidmay be precipitated from the solution by acidification and chilling.

The N-carbamyl amino acid is converted to an activated form such as amixed carbonic anhydride before it is reacted with 6-aminopenicillanicacid (6-APA) to yield the novel penicillins of this invention. Thisconversion is effected by dissolving the N-carbamyl amino,

acid in a ketone solvent containing a tri-(lower) alkyl amine andtreating the solution with anhydride forming reagent, eg a. lower alkylchloroformate or an aryl chloroformate at a temperature of from about 0to The final product:

from an acidified reaction mixture that has been saturated with ammoniumsulfate, or similar buffer.

An alternative method for producing the novel penicillins of theinvention is to couple the N-carbamyl amino acid.(produced as detailedabove) with 6-APA in an acetone-water (8:2) solution using acarbodiimide (e.g., dicyclohexyl-carbodiimide) as the condensingreagent. Excess carbodiimide is converted to the corresponding urea byacidification, the pH is adjusted to near neutrality and the urea isremoved by filtration. The products may be recovered by acidification orextraction as has been detailed above.

Other methods known to the art may be used such as coupling theN-carbamyl amino acid via its activated (e.g., p-nitro-phenyl) ester,imidazole carbonyl complex, azide, etc.

Still another alternative for forming the products of this invention(formula I) is to acylate 6-APA with an amino acid (formula ll), andreact the resulting penicillin with an alkali metal cyanate or analkaline earth metal cyanate in the presence of water to form the finalproduct (formula I). The acylation is carried out using procedures thatare conventional in the art.

It will be appreciated that certain of the compounds of this inventionexist in various states of solvation as well as in different opticallyactive forms. The various forms as well as their mixtures are within thescope of this invention. Ordinarily the new penicillin derived from theD-form of the amino acid is more active than that derived from theL-form or the DL-form.

The compounds of this invention have a broad spectrum of antibacterialactivity against both gram positive and gram negative organisms such asStaphylococcus aureus, Salmonella schottmuelleri, Pseudomonasaeruginosa, Proteus mirabilis, Escherichia coli and Streptococcuspyogenes. They may be used as antibacterial agents in a prophylacticmanner, e.g., in cleaning or disinfecting compositions, or otherwise tocombat infections due to organisms such as those named above, and ingeneral may be utilized in a manner similar to penicillin G and otherpenicillins. For example, a compound of formula I may be used in variousanimal species in an amount of about 1 to 200 mg./kg., daily, orally orparenterally, in single or two to four divided doses to treat infectionsof bacterial origin. By way of illustration the PD, orally in mice in asingle administration is of the order of 5.0 mg./kg. againstStreptococcus pyogenes.

Up to about 600 mg. of a compound of formula I or a physiologicallyacceptable salt thereof may be incorporated in an oral dosage form suchas tablets, capsules or elixirs or in an injectable form in a sterileaqueous vehicle prepared according to conventional pharmaceuticalpractice.

In cleaning or disinfecting compositions, e.g., in barns or dairyequipment, a concentration of about 0.0] to 1 percent by weight of suchcompounds admixed with, suspended or dissolved in conventional inert dryor aqueous carriers for application by washing or spraying may be used.They are also useful as nutritional supplements in animal feeds.

The following examples are illustrative of the invention.

EXAMPLE l 6-[ D- l carbamyl-2-pyrrolidinyl )formamido]- penicillanicacid Asuspension of l 15 mg of D-proline (1.0 mmole)'in 2.0 ml ofwateris treated with 146 mg of potassium cyanate (1.8 mmoles). Thesuspension is heated at about 80C for 5 minutes and the clear solutionis allowed to stand for 48 hours at room temperature with the pH beingadjusted to between 5.0 and 6.5 after 12, 24 and 36 hours with 6Nhydrochloric acid. At 48 hours the solution is chilled in an ice bathand acidification with hydrochloric acid to pH 1.0 precipitates the N-carbamyl derivative. This is washed with a few ml of cold water anddried over calcium chloride in a desiccator to yield 80 mg of thedesired compound.

A solution containing 79 mg of N-carbamyl-D- proline (0.5 mmoles) in 0.5ml of acetone containing .07 ml of triethylamine is converted to a mixedcarbonic anhydride by treating with 0.05 ml of ethyl chloroformate forabout 30 minutes at a temperature range of from 0C to 20C. A cold (about10C) solution of 108 mg of -aminopenicillanic acid (6-APA) in 2.0 ml of1:1 acetone-water containing 51 mg of triethylamine are added to thesolution of mixed anhydride and the reaction mixture stirred vigorouslyat about 0C for approximately 30 to 45 minutes. The volume of thesolution is reduced by evaporating the acetone at room temperature orbelow. Acidification precipitates6-[(D-l-carbamyl-2-pyrrolidinyl)formamido]- penicillanic acid.

EXAMPLE 2 6[(D-l-carbamyl-2-pyrrolidinyl)formamido1- penicillanic acidThe first part of the procedure of Example 1 is followed to obtainN-carbamyl-D-proline.

51.5 mg of N-carbamyl-D-proline are added to 6.0 ml of water, the pHadjusted to 6.5 with l.ON potassium hydroxide, and the final volumebrought to 7.0 ml with water. This is treated with a solution of l35 mgof dicyclohexylcarbodiimide in 28.0 ml of acetone. After 45 minutes at24C, mg of 6-APA is added with stirring and the reaction mixture setaside for 4 hours. The pH is readjusted to 6.5 with hydrochloric acidand the reaction mixture set aside for another 16 hours. The acetone isremoved in vacuo at 30C and the remaining aqueous mixture freed ofdicyclohexylurea and other insoluble material by filtration. Water isadded to the filtrate to adjust the volume to 10.0 ml, ammonium sulfateis added to saturate the solution, the pH is adjusted to 3.5 with 6Nhydrochloric acid and the solution then extracted five times with 20 mlportions of ethyl acetate. The ethyl acetate fractions are washed twotimes with 0.5 ml portions of H 0, pooled, dried with sodium sulfate andthen concentrated to dryness in vacuo at 30C. The concentrate is washedtwo times with 0.5 ml portions of benzene, the benzene insolublematerial is taken up in a few ml of acetone and the acetone solutiondried in vacuo at 30C to yield 30 mg of6-[(D-lcarbamyl-Z-pyrrolidinyl)formamido1-penicillanic acid.

EXAMPLE 3 6-[ D- l carbamyl-Z-pyrrolidinyl )formamido penicillanic acid,sodium salt One-half millimole of 6-[ D- l carbamyl-2-pyrrolidinyl)formamidol-penicillanic acid is dissolved in 5 ml of a 0.1Naqueous sodium hydroxide solution. Lyiophilization of the solutionyields the desired sodium sa t.

EXAMPLE 4 6-[ allo-D- l carbamyl-4-hydroxy-2-pyrrolidinyl)formamidol-penicillanic acid A suspension of l mmole ofhydroxy-D-proline(allo) in 2.0 ml of water is treated with 1.8 mmolespotassium cyanate. The suspension is heated at about 80C for fiveminutes and the clear solution is allowed to stand for 48 hours at roomtemperature with the pH being adjusted to between 5.0 and 6.5 after 12,.24 and 36 hours with 6N hydrochloric acid. At 48 hours the solution ischilled in an ice bath and acidification with hydrochloric acid to'pl-l-l.0 precipitates the N-carbamyl derivative. This is washed with a fewml of cold water and dried over calcium chloride in a desiccator.

A solution containing 0.5 mmoles of N-carbamylhydroxy-D-proline(a1lo) in0.5 ml of acetone containing 0.07 ml of triethylamine is converted to amixed carbonic anhydride by treating with 0.05 ml of ethyl chloroformatefor about 30 minutes at a temperature range of from C to 20C. A cold(about -l0C) solution of 0.5 mmoles of 6-aminopenicillanic acid (6- APA)in 2.0 ml of 1:1 acetone-water containing 51 mg of triethylamine isadded to the solution of mixed anhydride and the reaction mixturestirredvigorously at about 0C for approximately 30 to 45 minutes. Thevolume of the solution is reduced by evaporating the acetone at roomtemperature or below. Acidification precipitates 6-[ allo-D- lcarbamyl-4-hydroxy-2- pyrrolidinyl)formamido]penicillanic acid.

EXAMPLE 5 6-[(allo-D- l carbamyl-4-acetoxy-2pyrrolidinyl)-formamidol-penicillanic acid A suspension of 1 mmole ofhydroxy-D-proline (allo) in 2.0 ml of water is treated with 1.8 mmolesof potassium cyanate. The suspension is heated at about 80C for 5minutes and the clear solution is allowed to stand for 48 hours at roomtemperature with the'pH being adjusted to between 5.0 and 6.5 after 12,24, and 36 hours with 6N hydrochloric acid. At 48 hours the solution ischilled in an ice bath and acidification with hydrochloric acid to pH1.0 precipitates the N-carbamyl derivative. This is washed with a few'ml of cold water and dried over calcium chloride in a desiccator.

A solution of 50 mg of N-carbamyl-hydroxy-D- proline (allo) is dissolvedin 5.0 ml of pyridine, and 0.5 ml of acetic anhydride is added withstirring. After allowing the solution to set at room temperature forthree hours, 6.0N hydrochloric acid is added until a pH of 1.0 isattained. The acidified solution is extracted four times with 10.0 mlportions of ethyl acetate, the ethyl acetate fractions arepooled, washedtwo times with 4.0 ml portions of 0.1N hydrochloric acid, dried oversodium sulfate and evaporated to dryness in vacuo at 30C to obtainN-carbamyl-O-acetyl-D-proline (allo).

A solution containing 0.5 mmoles of Ncarbamyl-O-acetyl-D-proline (allo)in 0.5 ml of acetone containing 0.07 ml of triethylamine is converted toa mixed carbonic anhydride by treating with 0.05 ml of ethylchloroformate for about 30 minutes at a temperature range of from 0C to20C. A cold (about 10C) solution of 108 mg of 6-aminopenicillanic acidin 2.0 ml of 1:1 acetone-water containing 51 mg of triethylamine areadded to the solution of mixed anhydride and the reaction mixturestirred vigorously at about 0C for approximately 30 to 45 minutes. Thevolume of the solution is reduced by evaporating the acetone at roomtemperature or below. Acidification precipitates6-[(allo-D-1-carbamyl-4-acetoxy-2- pyrrolidinyl )formamido]penicillanicacid.

EXAMPLE 6 6-[( lCarbamy1-2-aziridinyl)formamido]-penicillanic acid Asuspension of 1 mmole of aziridine-2-carboxylic acid in 2.0 ml of wateris treated with 1.8 mmoles of potassium cyanate. The suspension isheated at about C for 5 minutes and the clear solution is allowed tostand for 48 hours at room temperature with the pH being adjusted tobetween 5.0 and 6.5 after 12, 24 and 36 hours with 6N hydrochloric acid.At 48 hours the solution is chilled in an ice bath and acidificationwith hydrochloric acid to pH 1.0 precipitates the N- carbamylderivative. This is washed with a few ml of cold water and dried overcalcium chloride in a desiccator.

A solution containing 0.5 mmole N-carbamylaziridine-Z-carboxylic acid in0.5 ml of acetone containing 0.07 ml of triethylamine is converted to amixed carbonic anhydride by treating with 0.05 ml of ethyl chloroformatefor about 30 minutes at a temperature range of from0C to 20C. A cold(about 1 0C) solution of 0.5 mmole of 6-aminopenicillanic acid (6- APA)in 2.0 ml of 1:1 acetone-water containing 51 mg triethylamine-is addedto the solution of mixed anhydride and the reaction mixture stirredvigorously at about 0C for approximately 30 to 45 minutes. The volume ofthe solution is reduced by evaporating the acetone at room temperatureor below. Acidification precipitates6-[(1carbamyl-Z-aziridinyl)-formamido]- penicillanic acid. 1

EXAMPLE 7 6-[( 1-Carbamyl-2-azetidinyl)formamido]-penicillanic acid Asuspension of l mmole azetidine-2-carboxylic acid in 2.0 ml of water istreated with 1.8 mmoles of potassium cyanate. The suspension is heatedat about 80C for five minutes and the clear solution is allowed to standfor 48 hours at room temperature with the pH being adjusted to between5.0 and 6.5 after 12, 24 and 36 hours with 6N hydrochloric acid. At 48hours the solution is chilled in an ice bath and acidification withhydrochloric acid to pH 1.0 precipitates the N- carbamyl derivative.This is washed with a few ml of cold water and dried over calciumchloride in a desiccator.

A solution containing 0.5 mmole of N-carbamylazetidine-Z-carboxylic acidin 0.5 ml of acetone containing 0.07 ml of triethylamine is converted toa mixed carbonic anhydride by treating with 005ml of ethyl chloroformatefor about 30 minutes at a temperature range of from 0C to 20C. A cold(about 10C) so lution of 0.5 mmole of 6-aminopenicillanic acid (6- APA)in 2.0 ml 1:1 acetone-water containing 51 mg triethylamine is added tothe solution of'mixed anhydride and the reaction mixture stirredvigorously at about 0C for approximately 30 to 45 minutes. The volume ofthe solution is reduced by evaporating the acetone at room temperatureor below. Acidification precipitates 6-1carbamyl-Z-azetidinyl)formamido1-penicillanic acid.

EXAMPLE 8 6-[( l-Carbamyl-2-hexahydroazepinyl)formamido1- penicillanicacid A suspension of l mmole of hexahydroazepine-Z- carboxylic acid in2.0 ml of water is treated with 1.8 mmoles of potassium cyanate. Thesuspension is heated at about 80C for 5 minutes and the clear solutionis allowed to stand for 48 hours at room temperature with the pH beingadjusted to between 5.0 and 6.5 after 12, 24 and 36 hours with 6Nhydrochloric acid. At 48 hours the solution is chilled in an ice bathand acidification with hydrochloric acid to pH 1.0 precipitates theN-carbamyl derivative. This is washed with a few ml of cold water anddried over calcium chloride in a desiccator.

A solution containing 0.5 mmole N-carbamylhexahydroazepine-Z-carboxylicacid in 0.5 ml of acetone containing 0.07 ml of triethylamine isconverted to a mixed carbonic anhydride by treating with 0.05 ml ofethyl chloroformate for about 30 minutes at a temperature range of fromC to -20C. A cold (about l0C) solution of 0.5 mmole of6-aminopenicillanic acid (6-APA) in 2.0 ml of 1:1 acetone-watercontaining 51 mg triethylamine is'added to the solution of mixedanhydride and the reaction mixture stirred vigorously at about 0C forapproximately 30 to 45 minutes. The volume of the solution is reduced byevaporating the acetone at room temperature or below. Acidificationprecipitates 6-[( l-carbamyl-2-hexahydroazepinyl)formamido[-penicillanicacid.

EXAMPLE 9 6-[( l-Carbamyl-4-hydroxy-piperidinyl-2)formamido]-penicillanic acid A suspension of l mmole 4-hydroxypipecolic acid in 2.0ml of water is treated with 1.8 mmoles of potassium cyanate. Thesuspension is heated at about 80C for 5 minutes and the clear solutionis allowed to stand for 48 hours at room temperature with the pH beingadjusted to between 5.0 and 6.5 after 12, 24 and 36 hours with 6Nhydrochloric acid. At 48 hours the solution is chilled in an ice bathand acidification with hydrochloric acid to pH 1.0 precipitates theN-carbamyl derivative. This is washed with a few ml of cold water anddried over calcium chloride in a desiccator.

A solution containing 0.5 mmole N-carbamyl-4- hydroxy-pipecolic acid in0.5 ml of acetone containing 0.07 ml of triethylamine is converted to amixed carbonic anhydride by treating with 0.05 ml ethyl chloroformatefor about 30 minutes at a temperature range of from 0C to -20C. A cold(about -l0C) solution of 0.5 mmole of 6-amino-penicillanic acid (6-APA)in 2.0 ml of 1:1 acetone-water containing 51 mg of triethylamine isadded to the solution of mixed anhydride and the reaction mixturestirred vigorously at about 0C for approximately 30 to 45 minutes. Thevolume of the solution is reduced by evaporating the acetone at roomtemperature or below. Acidification precipitates 6[(lcarbamyl-4-hydroxy-piperidinyl-Z)formamidolpenicillanic acid.

EXAMPLE l0 6-[(l-Carbamyl-5-hydroxy-piperidinyl-2)-formamido]penicillanic acid Asuspension of l mmole of S-hydroxypipecolic acid in 2.0 of water istreated with 1.8 mmoles of potassium cyanate. The suspension is heatedat about C for 5 minutes and the clear solution is allowed to stand for48 hours at room temperature with the pH being adjusted to between 5.0and 6.5 after 12, 24 and 36 hours with 6N hydrochloric acid. At 48 hoursthe solution is chilled in an ice bath and acidification withhydrochloric acid to pH 1.0 precipitates the N-carbamyl derivative. Thisis washed with a few ml of cold water and dried over calcium chloride ina desiccator.

A solution containing 0.5 mmole N-carbamyl-S- hydroxy-pipecolic acid in0.5 ml of acetone containing 0.07 ml of triethylamine is converted to amixed carbonic anhydride by treating with ethyl chloroformate for about30 minutes at a temperature range of from 0C to -20C. A cold (about l0C)solution of 0.5 mmole of 6-aminopenicillanic acid (6-APA) in 2.0 ml of1:1 acetone-water containing 51 mg triethylamine is added to thesolution of mixed anhydride and the reaction mixture stirred vigorouslyat about 0C for approximately 30 to 45 minutes. The volume of thesolution is reduced by evaporating the acetone at room temperature orbelow. Acidification precipitates 6-[( lcarbamyl-5-hydroxy-piperidinyl-2)formamido1- penicillanic acid.

What is claimed is:

l. A compound of the formula wherein R is selected from the groupconsisting of hydrogen, lower alkyl,

an alkali metal and an alkaline earth metal; wherein R is selected fromthe group consisting of hydrogen, hydroxy, and

wherein R is selected from the group consisting of lower alkyl andmonocyclic carbocyclic aryl; wherein nisOor l,mis0, 1,2, 3,or4,pis 1,2,3,0r4andthe sum ofn+m+pis l, 2, 3, 4, or 5.

2. A compound as defined in claim 1 wherein R is hydrogen.

3. A compound as defined in claim 1 wherein R is hydroxy.

4. A compound as defined in claim 1 wherein R is 5. A compound asdefined in claim 1 wherein n is l, m is l, andpis l.

6. A compound as defined in claim 5 having the name 6-[ D- 1-carbamyl-2-pyrrolidinyl )formamido]- penicillanic acid.

7. A compound as defined in claim 5 having the name 6- allo-D- l-carbamyl-4-hydroxy-2-pyrrolidinyl )formamido]-penicillanic acid.

1. A COMPOUND OF THE FORMULA
 2. A compound as defined in claim 1 whereinR1 is hydrogen.
 3. A compound as defined in claim 1 wherein R1 ishydroxy.
 4. A compound as defined in claim 1 wherein R1 is
 5. A compoundas defined in claim 1 wherein n is 1, m is 1, and p is
 1. 6. A compoundas defined in claim 5 having the name6-((D-1-carbamyl-2-pyrrolidinyl)formamido)-penicillanic acid.
 7. Acompound as defined in claim 5 having the name6-((allo-D-1-carbamyl-4-hydroxy-2-pyrrolidinyl)formamido)-penicillanicacid.